Compartmentalized proteases contain proteolytic sites sequestered from the rest of the cellular environment. Inserting a folded protein into the proteolytic chamber of such proteases is an energy-dependent process with steps that include recognition, unfolding and progressive advancement into the proteolytic chamber. ATPase subunits of regulatory complexes associated with these proteases produce the required force. We will examine the processes of substrate unfolding and translocation of the 26S proteasome, the major cytoplasmic and nuclear protease of the eukaryotic cell. Specifically, we intend to investigate the mechanism by which the ATPase subunits of the 19S regulatory complex utilize energy derived from ATP to drive these events. We have developed proteasome substrates whose properties facilitate examining the interactions between force production and consumption. Our recent findings lay the foundation for a novel approach to studying how proteasomes produce and consume energy. In these projected studies we will create a series of substrates that offer diverse and calibrated challenges to proteasome function, develop relevant assays to measure how well the proteasome meets these challenges, and examine the capacity of proteasome subassemblies and mutants to perform these tasks. Specific Aim 1. Test the properties of the interaction between sites of energy production and unfolding. Specific Aim 2. Develop alternate assays of substrate unfolding and fate and use these to test the function of proteasome components. These observations will be further extended by developing and applying biochemical assays for proteasome-mediated unfolding. Specific Aim 3. Examine genetic interactions affecting insertional arrest. Site-directed mutations of conserved ATPase motifs will be made to abrogate ATP hydrolysis, ATP binding, communication between ATPases and substrate engagement. Random mutations of additional 19S proteins will be made and tested. [unreadable] [unreadable]